PRDM1 silences stem cell-related and inhibits proliferation of human colon tumor organoids

Changlong Liua, Carolyn E. Banistera, Charles C. Weigea, Diego Altomarea, Joseph H. Richardsonb, Carlo M. Contrerasb, and Phillip J. Buckhaultsa,1

aDepartment of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, SC 29208; and bDivision of Surgical Oncology, Department of Surgery, The University of Alabama at Birmingham, Birmingham, AL 35233

Edited by Kenneth W. Kinzler, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD, and approved April 27, 2018 (received for review February 21, 2018) PRDM1 is a tumor suppressor that plays an important role in B and of genes encoding c-, Spi-B, Id3, and Pax-5 (8). In human lymphomas. Our previous studies demonstrated that and mouse cells PRDM1 target genes include IFN-β, , IL- PRDM1β is a -response in human colorectal cancer cells. 2, c-Fos, Dusp16, and TP53 (1, 7, 13, 20, 22). However, the function of PRDM1β in colorectal cancer cells and PRDM1 interacts with the p53 tumor-suppressor pathway, and colon tumor organoids is not clear. Here we show that PRDM1β its ability to repress genes indicates it may have an important role is a p53-response gene in human colon organoids and that low as a tumor suppressor (23). In HCT116 cells, PRDM1 regulates PRDM1 expression predicts poor survival in colon cancer patients. cell growth through the repression of p53 , and We engineered PRDM1 knockouts and overexpression clones in p53 is able to activate PRDM1 expression by binding to an al- RKO cells and characterized the PRDM1-dependent transcript land- ternative promoter (22). Our previous study has shown that scapes, revealing that both the α and β transcript isoforms repress PRDM1 is activated by specific polymorphic variants of p53 in MYC-response genes and stem cell-related genes. Finally, we show RKO colon cancer cells (24). Also a recent study showed that that forced expression of PRDM1 in human colon cancer organoids PRDM1can inhibit SW620 colon cancer cell proliferation by prevents the formation and growth of colon tumor organoids in inhibiting c-Myc (25). The mechanisms by which PRDM1 acts on

vitro. These results suggest that p53 may exert tumor-suppressive human colon cells is incompletely understood and may lead to GENETICS effects in part through a PRDM1-dependent silencing of stem cell insights relevant to colon cancer and normal intestinal stem cell genes, depleting the size of the normal intestinal stem cell com- maintenance. In this study, we knocked out and overexpressed partment in response to DNA damage. PRDM1 in RKO colon cancer cells and in colon cancer orga- noids to address the roles of PRDM1 and its target gene land- TP53 | PRDM1 | colorectal cancer | organoids | CRISPR scape in colon cancer cell proliferation. We show that PRDM1 activates and represses a large number of target genes related to RDM1 is a positive regulatory domain , proliferation and differentiation and that it powerfully inhibits Pwhich is also known as “B lymphocyte-induced maturation clonogenic survival of primary colon tumor organoids. protein” (Blimp-1). It was first identified as a protein that binds Results to and represses expression from the IFN-β promoter (1). It is a master regulator of the differentiation of plasma cells from B PRDM1 Is a p53-Responsive Gene in Normal Colon Organoids and Is PRDM1 cells, driving differentiation (2). PRDM1 plays crit- Correlated with Disease-Free Survival in Colon Cancers. The α ical roles in the differentiation and development of many other gene encodes a long, 5,164-bp transcript (PRDM1 )anda types of cells in mouse and in other model organisms (3–7). PRDM1 induces maturation by affecting genes that are Significance critical for mature B cell identity and cellular proliferation (8– 10). In T cells, PRDM1 is expressed in both memory and effector Our previous studies demonstrated that PRDM1β is activated by cell types, and it regulates the of these two pop- p53 accumulation in human colorectal cancer cells. However, the ulations (11, 12). PRDM1 is induced by IL-2, which is itself function of PRDM1β in colorectal cancer cells and colon tumor regulated by PRDM1 through the binding of PRDM1 to the IL- organoids is not clear. Here we show that PRDM1β is a p53-response 2 promoter. This auto feedback forms a regulation loop in T cells gene in human colon organoids and that low PRDM1 expression (13, 14). In the early developing mammalian embryo, PRDM1 predicts poor survival in colon cancer patients. Also, PRDM1α and controls global epigenetic changes that are required for specifi- PRDM1β repress a largely overlapping suite of genes, many cation of the primordial germ cells (15–17). In primordial germ of which are stem cell-related genes. Moreover, we show that cells, PRDM1 associates with PRMT5, an arginine-specific his- forced expression of PRDM1β prevents the proliferation of colon tone methyl transferase, and induces the dimethylation of arginine tumor organoids. This work provides support for a role of PRDM1β 3 of the histone H2A and H4 tails. The PRDM1 and PRMT5 in regulating normal colon stem cell proliferation. complex is implicated in repressing genes associated with a so- Author contributions: C.L. and P.J.B. designed research; C.L., C.E.B., C.C.W., D.A., J.H.R., matic cell program, thus maintaining the primordial germ cells in and C.M.C. performed research; J.H.R. and C.M.C. contributed new reagents/analytic an undifferentiated state (18). tools; C.L. and P.J.B. analyzed data; and C.L. and P.J.B. wrote the paper. PRDM1 is a transcriptional with mechanisms that The authors declare no conflict of interest. vary in a context-dependent manner. PRDM1 has DNA-binding This article is a PNAS Direct Submission. activity and the ability to recruit diverse chromatin-modifying Published under the PNAS license. proteins, such as G9a, HDAC1, HDAC2, and the transcriptional Data deposition: The data reported in this paper have been deposited in the Gene Ex- corepressor Groucho, generating regulatory complexes that can pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. silence specific genes during cell differentiation (19–21). GSE101668). PRDM1 recruits the G9a methyltransferase through the first two 1To whom correspondence should be addressed. Email: [email protected]. zinc fingers, thus leading to H3K9 methylation and transcrip- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tional silencing (20). PRDM1 target genes have been reported in 1073/pnas.1802902115/-/DCSupplemental. several tissues. In plasma cells PRDM1 represses the expression

www.pnas.org/cgi/doi/10.1073/pnas.1802902115 PNAS Latest Articles | 1of10 Downloaded by guest on September 28, 2021 shorter 4,675-bp transcript (PRDM1β) (Fig. 1A). The two tran- PRDM1 regulation in human primary colon cells, we cultured scripts differ in transcriptional start sites and in the lengths of human normal colon organoids named “P08182015” (Table 1) in their coding sequences, with PRDM1β encoding a 727-aa pro- 3D culture (Fig. 1B) (28–30) that contained heterogeneous cell tein that is 102 aa shorter than the larger protein encoded by populations, reflecting the in vivo cell-type diversity. We acti- PRDM1α. The short-transcript isoform contains a unique 5′ vated the p53 protein (Fig. 1D) by treating the organoids with UTR, starting from a transcriptional start site located within either the MDM2 inhibitor nutlin3a (31) or the DNA-damaging intron 3. The relative activities of these two isoforms are not agent etoposide. Both GDF15, a known p53-response gene (24), understood, although some results suggest that PRDM1β en- and PRDM1β, but not PRDM1α (SI Appendix, Fig. S1B), were codes an inactive or less active polypeptide (26). Overall the significantly increased after exposure to nutlin3a or etoposide expression of both PRDM1 isoforms is low in most colon can- (Fig. 1 C and D). cer cell lines. We confirmed this by examining GSE46549 data We reasoned that p53 mutation could lead to the reduction (SI Appendix, Fig. S1A) (27). We and others have previously of PRDM1 expression in colorectal tumors. To assess this, we shown that PRDM1β is activated by p53 (22, 24). To explore analyzed the expression of PRDM1 in 222 colorectal cancer

Fig. 1. PRDM1 is a p53-responsive gene in human normal colon organoids and is correlated with disease-free survival in colon cancers. (A) Schematic representation of two transcript isoforms (PRDM1α and PRDM1β) derived from the human PRDM1 gene. (B) Morphology of a human normal colon organoid culture in vitro. (Scale bars: 2 mm.) (C) Organoids were treated with either nutlin3a or etoposide, and PRDM1β expression was evaluated by qPCR. The GDF15 expression level was determined as control. Data are shown as the mean ± SEM of technical triplicates. *P < 0.05, **P < 0.01. (D) PRDM1β protein and p53 levels were determined by Western blot on normal colon organoids that were treated with nutlin3a for 48 h. (E) The boxplot shows PRDM1 expression in colorectal tumors in the context of TP53-WT (n = 110) or TP53-Mutation (n = 112) from the TCGA database. The y axis is log2 scale of expression. Each dot represents one patient. (F) The Kaplan–Meier survival plots were obtained using the R2 Genomics Analysis and Visualization Platform (https://hgserver1.amc. nl/cgi-bin/r2/main.cgi) and display the probability of disease-free survival of 209 colon cancer patients: high PRDM1 >212.9, n = 176; low PRDM1 <212.9, n = 33. (Left) and 320 colon cancer patients: high PRDM1 >227.6, n = 272; low PRDM1 <227.6, n = 48. (Right). (G) Patient survival and PRDM1β expression in- formation was obtained from Xena Functional Genomics Explorer. The Kaplan–Meier survival plot was generated using R2 Kaplan Meier Scanner and displays the probability of disease-free survival of 357 colon cancer patients (PRDM1β expression range: 0 to ∼2.79; high PRDM1 >1.345, n = 25; low PRDM1 <1.345, n = 332). P values on the plots are from the log-rank test for the comparisons of the low and high PRDM1 expression groups.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1802902115 Liu et al. Downloaded by guest on September 28, 2021 Table 1. Organoid information used in this study Name Patient age, y Patient gender Stage TP53 status Organoid type

P08182015 57 Female IV Wild type Normal P10152015 56 Male IIA Mutant Tumor P07132016 62 Male IIA Mutant Tumor

patients from The Cancer Genome Atlas (TCGA) COAD- except in the p53-KO RKO cells, and PRDM1β was activated READ samples for which both PRDM1 expression and after nutlin3a treatment in wild-type RKO cells. However, PRDM1β TP53 mutation data were available. PRDM1 expression in is not activated in p53-KO RKO cells or in PRDM1-KO RKO TP53 wild-type colorectal tumors is significant higher than found cells after nutlin3a treatment (SI Appendix, Fig. S2D). in TP53-mutant colorectal tumors (Fig. 1E). These data showed We next generated PRDM1α- and PRDM1β-overexpressing that PRDM1β is a p53-regulated gene in primary human normal (OE) derivatives of RKO colon cancer cells. We cloned colon organoids and colorectal tumors. Next, we examined PRDM1α and PRDM1β cDNA into a lentiviral vector which PRDM1 in published datasets with clinical encodes a GPF marker polypeptide linked to the PRDM1 gene outcome information and noted that the loss of PRDM1 ex- product by the P2A peptide sequence. We also generated a pression is correlated with poor survival in B cell lym- control vector expressing only GFP (Fig. 2H). We produced phoma (SI Appendix, Fig. S1C) and in colon adenocarcinoma replication-defective viral particles in the 293T packaging line (Fig. 1 F and G). and infected RKO cells at a multiplicity of infection (MOI) of 3.0 to produce pools of PRDM1α-, PRDM1β-, and GFP-OE + Generation and Characterization of PRDM1-KO and PRDM1- cells. Three days after infection, we isolated the GFP cells by Overexpressing RKO Cell Lines. To evaluate the roles of PRDM1 FACS. The PRDM1α-OE and PRDM1β-OE cells were viable in colon cancer cells, first we generated PRDM1-KO deriva- and exhibited morphology similar to that of GFP-OE cells (Fig. tives in RKO colon cancer cells by employing CRISPR/ 2I). PRDM1α-OE and PRDM1β-OE cells showed high levels of Cas9 technology. We developed a strategy that used two small PRDM1α and PRDM1β transcript (Fig. 2J) and protein ex- guide RNAs (sgRNAs) to create a deletion of the intervening pression (Fig. 2K). GENETICS segment by inducing two double-strand breaks (DSBs) into ge- nomic DNA (Fig. 2A) (32, 33). We designed eight sgRNAs PRDM1α and PRDM1β Have Similar Response Genes in RKO Cells. The and synthesized them as gBlock fragments containing the PRDM1 transcript landscape in colon cancer cells is not yet U6 promoter and the guide RNA (gRNA) targeting and scaffold described nor are the relative contributions of the PRDM1α and sequence (Fig. 2B). The Cas9-GFP plasmid (34) and sgRNA -β isoforms to PRDM1-response gene expression. To identify pairs were simultaneously introduced into cells and then were the transcriptional differences between PRDM1-KO cells and subjected to FACS based on GFP (Fig. 2C). We tested the PRDM1α- and PRDM1β-OE RKO cells, we performed RNA- abilities of several pairs of sgRNAs to induce PRDM1 genomic sequencing (RNA-seq) experiments on the cell lines listed in SI deletions large enough to detect by sizing PCR products span- Appendix, Fig. S3A. We obtained more than 30 million uniquely ning the deleted regions (SI Appendix, Fig. S2A). The ability to mapped reads for each sample (SI Appendix, Fig. S3B), with the create a genomic deletion was tested by conventional PCR using two biological replicates of each sample being highly reproduc- primer pairs located outside the sgRNAs’ binding region (SI ible (SI Appendix,Fig.S3C). First, we evaluated the global Appendix, Table S1). We obtained individual KO clones that changes in gene expression induced by PRDM1α overexpression knocked out only PRDM1α (sgRNA1–sgRNA2 and sgRNA3– and PRDM1β overexpression compared with PRDM1 KO. We sgRNA4) (SI Appendix, Fig. S2B), clones that knocked out only found that the set of genes regulated by PRDM1α was highly PRDM1β (sgRNA5–sgRNA7 and sgRNA6–sgRNA7) (SI Ap- similar to that regulated by PRDM1β (Fig. 3A). We observed pendix, Fig. S2C), and clones that knocked out both PRDM1α 2,820 genes that were significantly different [false discovery rate and PRDM1β (sgRNA1–sgRNA8) (Fig. 2 D and E). We ana- (FDR) adjusted P < 0.05] between PRDM1-KO and either lyzed 24 randomly selected individual clones potentially knocked PRDM1α-OE or PRDM1β-OE cells. Surprisingly, over half of out for both PRDM1α and PRDM1β by PCR with primers these genes (1,625) were significantly different in both the flanking the disrupted region (Fig. 2D, outer primers indicated PRDM1α and the PRDM1β isoforms (FDR adjusted P < 0.05) by red arrows) and noted the presence of at least one allele of an (Fig. 3B). We identified 925 genes that were up-regulated and abnormal sized PCR product in 18 samples (75%) (Fig. 2E). The 727 genes that were down-regulated by both PRDM1α and absence of both alleles of the intervening deleted region was PRDM1β (FDR adjusted P < 0.05) (Fig. 3C). The genes that confirmed by PCR using primers completely contained within were repressed by both PRDM1α and PRDM1β included MYC the removed region flanking exon 2 (Fig. 2D, inner primers in- and ID3, previously reported in B cells (8), and CDK6 and JAG1. dicated by green arrows). Of the 18 clones with at least one Interestingly, we found that members of JUN family—JUN, disrupted allele, we noted five clones that were completely de- JUNB, and JUND—were activated by PRDM1. Unsupervised void of exon2 sequences. (Fig. 2E, Lower). We then randomly hierarchical cluster analysis shows high concordance between the selected two clones with disrupted PRDM1α and PRDM1β for replicate cell lines for both PRDM1α and PRDM1β expression, further characterization by cell proliferation. PRMD1 disrup- as well as in two broad classes of genes, those that are repressed tion had no noticeable effect on RKO cell morphology (Fig. 2F) and those that are activated (Fig. 3D). These results indicate that or on cell proliferation as assayed by 5-ethynyl-2′-deoxyuridine PRDM1α and PRDM1β may have a similar function in the (EdU) imaging (Fig. 2G). These results demonstrated a ro- regulation of cell biology. bust method to produce genomic deletions using CRISPR/ Cas9 technology and that the PRDM1 gene is not necessary for PRDM1 Arrested MYC Transcriptional Programs by Inhibiting MYC the survival of RKO colon cancer cells in vitro. Then we treated Gene Expression. To examine the possible biological influences TP53 wild-type RKO, TP53-KO RKO, PRDM1 wild-type RKO, of gene networks regulated by PRDM1 in colon cancer cells, we and PRDM1-KO RKO cells with nutlin3a or with DMSO as a rank ordered all expressed genes by their expression ratio in control. The result showed that p53 was activated by nutlin3a PRDM1-OE versus PRDM1-KO cells and performed unbiased

Liu et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 28, 2021 Fig. 2. Generating PRDM1-KO and PRDM1-OE RKO cell lines. (A) Schema of the strategy using two sgRNAs and CRISPR/Cas9. Two sgRNAs (sgRNA-A and sgRNA-B) were designed to remove a piece of DNA from the PRDM1 gene. Two DSBs were induced by transient expression of sgRNA-A and sgRNA-B plus CRISPR/Cas9. (B) Small synthetic genes (455 bp) encoding sgRNAs were synthesized as gBlocks containing the U6 promoter, the gRNA targeting region, and the scaffold sequence. (C) The procedure for generating KO clones. (D) Schematic illustration of the PRDM1 gene locus, sgRNAs, and screening primer lo- cations. About 13 kb of the DNA sequence between gRNA1 and gRNA8 was deleted. (E) PCR screening of individual clones. Clones positive for outer and negative for inner PCR products were considered null for both PRDM1 alleles. PRDM1-KO-2 and PRDM1-KO-5 clones are shown. (F) Morphology of PRDM1 wild-type RKO cells and PRDM1-KO RKO cells. (Scale bars: 200 μm.) (G) Cell proliferation was visualized by the incorporation Edu (orange); the counterstain was Hoechst (blue). (Scale bars: 200 μm.) (H) Schematic illustration of lentiviral vectors used for PRDM1 overexpression. The GFP-only vector was constructed as a control. (I) Morphology of GFP-only, PRDM1α-OE, and PRDM1β-OE RKO cell lines. (Scale bars: 200 μm.) (J) Heatmap of PRDM1 exon expression derived from RNA-seq reads. (K) PRDM1α and PRDM1β protein levels were determined by Western blot.

gene set enrichment analysis (GSEA) using the Hallmark dataset colon cancer cells (Fig. 4A). PRDM1 is known to regulate IFN from the Molecular Signatures Database (MSigDB) (35). We genes, as it was first isolated as a repressor of the IFN-β pro- characterized the transcriptional consequences of PRDM1 in moter (1). We observed that IFN-response genes were strongly

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Fig. 3. Global differential gene expression of PRDM1-KO and PRDM1-OE RKO cells. (A) Volcano plots comparing differential gene-expression levels between PRDM1-KO and PRDM1α-OE RKO cells and between PRDM1-KO and PRDM1β-OE RKO cells. Red dots show up-regulated genes, and blue dots show genes that are repressed in PRDM1-OE cells compared with PRDM1-KO cells (FDR P < 0.05). (B) Venn diagrams display the gene set of overlap between PRDM1α-and PRDM1β-regulated genes compared with PRDM1-KO. (C) The scatter plot shows 925 genes up-regulated and 727 genes down-regulated by both PRDM1α and PRDM1β.(D) Heat map showing common differentially expressed genes in each replicate.

repressed by PRDM1 in RKO colon cancer cells (SI Appendix, and B) and tested cell-cycle distributions (SI Appendix, Fig. S5 Fig. S4A). PRDM1 was initially described as a master regulator C–E). These results suggested that PRDM1 could regulate the of B cell differentiation, repressing ID3, NFIX, PSMB10, PSM8, cell cycle. Then we tested the proliferation rates of RKO de- SP110, and TAPBP (36). We found that these B cell target genes rivatives. PRDM1 engineering had a minimal effect on RKO cell were also highly repressed by PRDM1 in RKO colon cancer cells growth (SI Appendix, Fig. S5F). Therefore we evaluated the (SI Appendix, Fig. S4B). Among all gene sets in the Hallmark function of PRDM1 in human colon tumor organoids grown in dataset, MYC targets were significantly enriched among the 3D culture. To this end, we isolated and established two human PRDM1 down-regulated genes (Fig. 4B). Consistent with pre- colon tumor organoid lines from clinical surgery specimens, vious reports in HCT116 cells (22), we observed that MYC which we named “P07132016” and “P10152015” (Table 1). mRNA levels negatively correlated with PRDM1 expression These organoid lines could proliferate for over a year without (Fig. 4C), which we confirmed in RKO cells by real-time qPCR WNT3a in 3D culture in vitro (SI Appendix, Fig. S6 A and B). (Fig. 4D) and Western blot (Fig. 4E). These results indicate that We checked PRDM1β expression levels in these organoids PRDM1 may inhibit cell proliferation by inhibiting the MYC and found the expression level of PRDM1β was low in transcriptional program. P10152015 and was undetectable in P07132016 (SI Appendix, Fig. S6C). Because p53 protein activation results in PRDM1β PRDM1 Inhibits Tumor Organoid Formation in 3D Culture. It has been transcript activation, we overexpressed PRDM1β in these human reported that reconstitution of PRDM1 leads to impaired G2/M tumor organoids by lentiviral infection (SI Appendix, Fig. S6C) + − cell-cycle progression in PRDM1-null NK cell lines (23). We and collected GFP and GFP cells at different time points by performed GSEA on the entire set from the cell sorting. We then performed an organoid-forming assay on + − target gene signature from the MSigDB (SI Appendix, Fig. S5 A PRDM1 and PRDM1 cells (Fig. 5A). We first noted that

Liu et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 28, 2021 Fig. 4. PRDM1 inhibits MYC and MYC target genes. (A) Volcano plot of GSEA of the MSigDB Hallmark database. The FDR versus the normalized enrichment score (NES) for each evaluated gene set is shown. Blue dots are significantly enriched gene sets (FDR adjusted P value <0.05). (B) GSEA of MYC target genes, rank ordered by ANOVA T statistic. (C) Relative expression levels of MYC in RKO cells by RNA-seq. (D) mRNA levels determined by qPCR in PRDM1-KO and PRDM1-OE RKO cells. Data are shown as the mean ± SEM of technical triplicates. *P < 0.05, **P < 0.01. (E) Myc protein levels in PRDM1-OE and PRDM1-KO RKO cells were determined by Western blotting.

PRDM1β infection had a dramatic negative impact on cell testinal stem cell signature in colon cancer cells (Fig. 6A). These numbers in both P07132016 (Fig. 5B) and P10152015 organoids results indicate that forced expression of PRDM1β in colon tumor + (Fig. 5D). The percentage of PRDM1 cells dropped from day organoids prevents cancer stem cell proliferation and expansion, 2 to day 20 compared with control GFP cells, which increased possibly through silencing of the stem cell gene-expression + − during the same period (Fig. 5 C and E). GFP and GFP cells network. were obtained from the PRDM1β and control lentivirus infec- tions and were plated to assess organoid-formation abilities. Discussion PRDM1-expressing cells proliferated very slowly in P07132016 We have investigated the role of PRDM1 in both RKO colon and formed only very small organoids compared with control cancer cells and human colon tumor organoids by PRDM1-KO − cells. However, GFP cells from the same transduction pro- and PRDM1 overexpression. We developed a strategy of using liferated very well and formed regular tumor organoids (Fig. 5 F two sgRNAs to efficiently create a deletion of the intervening and G and SI Appendix, Fig. S6E). In P10152015 organoids, the segment by inducing two DSBs into genomic DNA by employing PRDM1-expressing cells were viable but did not proliferate or CRISPR/Cas9 technology (40, 41) using synthesized gBlocks for expand into organoids in 3D culture (Fig. 5 H and I and SI sgRNA production. We knocked out the PRDM1 gene and Appendix, Fig. S6E). We also knocked out PRDM1 in the two overexpressed PRDM1α and -β in RKO colon cancer cells and organoids P10152015 and P07132016 and performed organoid- found that PRDM1 can repress the expression of stem cell- forming assays in 3D cultures. The result showed that there was related genes. Overexpressing PRDM1α and PRDM1β in RKO no significant difference between control sgRNA and PRDM1 cells showed similar effects on gene regulation. Since PRDM1 is sgRNA (SI Appendix, Fig. S6F), probably due to the low level of described as a repressor in B and T cells, the up-regulated PRDM1 expressed in both tumor organoids. 925 genes might be regulated by PRDM1 indirectly. Interest- Previous studies have shown an embryonic stem cell-like gene- ingly, Jun family members such as JUN, JUNB, and JUND were expression signature in breast cancer (37) and in poorly differ- up-regulated by PRDM1 (Fig. 3C). It is noteworthy that KDM5B entiated aggressive human tumors (38). Also, an intestinal stem expression levels were increased by PRDM1 overexpression, cell signature could identify colorectal cancer stem cells (39). which could lead to H3K4me2 and H3K4me3 demethylation and Therefore we performed GSEA analysis using RNA-seq data in gene repression through indirect mechanisms. PRDM1 can re- RKO cells on these signatures and found PRDM1 could result in press specific genes not only by recruiting diverse chromatin- the down-regulation of embryonic stem cell signature and in- modifying proteins, such as G9a, HDAC1, HDAC2, and the

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Fig. 5. PRDM1 inhibits colon tumor organoid clonal survival. (A) Schematic of the tumor organoid-forming assay. (B) Flow cytometry scatter plots showing the + + percentage of GFP cells in P07132016 tumor organoids at the different time points indicated. (C) Percentages of GFP cells were normalized to day 2 starting + material. Relative GFP ratios from patient P07132016 are shown. (D) Flow cytometry scatter plots showing the percentage of GFP cells in P10152015 tumor organoids + at the indicated time points. (E) Percentages of GFP cells were normalized to day 2 starting material. The relative GFP ratios from patient P10152015 are shown. + − (F) Representative images of the organoid-forming assay from patient P07132016 colon tumor organoids. GFP cells and GFP cells from control or PRDM1-infected organoids were plated in Matrigel at a density of 500 cells per well in 24-well plates and were cultured for 14 d. Representative images are shown. (Magnification, 40×.) (G) Patient P07132016 organoids from control and PRDM1-infected cells were GPF+. Organoids infected with control virus were larger and greater in number than those infected with PRDM1β virus. (H) Representative images of the organoid-forming assay from patient P10152015 colon tumor organoids. GFP+ cells and GFP− cells from control or PRDM1-infected organoids were plated in Matrigel at a density of 500 cells per well in 24-well plates and were cultured for 3–5 wk. Repre- sentative images are shown. (Magnification, 40×.) (I) Organoids derived from patient P10152016 infected with lentivirus containing PRDM1β and control virus are + GPF . Organoids infected with control virus are larger and greater in number than those infected with PRDM1β virus. (Scale bars in G and I:200μm.)

Liu et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 28, 2021 transcriptional corepressor Groucho, generating regulatory PRDM1 KO with CRISPR/Cas9. Human codon-optimized Streptococcus pyogenes complexes (19–21), but also by up-regulating demethylase genes wild-type Cas9 (Cas9-2A-GFP) was obtained from Addgene (no. 44719). Chi- such as KDM5B. meric guide RNA expression cassettes with different sgRNAs (sgRNA1: Two previous studies of the role of PRDM1 in cell proliferation AGCCGCACAGACGCGCACCT; sgRNA2: AAAACGTGTGGGTACGACCT; sgRNA3: CACAGGAACGGCGGGACAAT; sgRNA4: TGATGGCGGTACTTCGGTTC; sgRNA5: in HCT116 and SW620 colon cancer cells draw different, con- GCCATAACAAAGCGAACACT; sgRNA6: GTGTTACTTTAGGACTTGGA; sgRNA7: tradictory conclusions (22, 25). PRDM1 loss of function in these GCAGAAATCAGGGCGGAAAC; sgRNA8: AGGGGCAGAACCGACATTAC) were studies was based on knockdown technology in which residual ordered as gBlocks. These gBlocks were amplified by PCR using the following PRDM1 expression could still remain if PRDM1 is maintained at primers: gBlock_Amplifying_F: 5′-GTACAAAAAAGCAGGCTTTAAAGG-3′ and a low level in RKO cells (SI Appendix, Figs. S1A and S3 D and E). gBlock_Amplifying_R: 5′-TAATGCCAACTTTGTACAAGAAAGC-3′. The PCR We used PRDM1-KO technology to completely abate PRDM1 product was purified by Agencourt Ampure XP PCR Purification beads gene expression. Our study found that neither PRDM1-KO nor according to the manufacturer’s protocol (Beckman Coulter). One micro- gram of Cas9 plasmid and 0.3 μg of each gRNA gBlock were cotransfected PRDM1 OE could affect RKO colon cancer cell proliferation + significantly (SI Appendix,Fig.S5). The different results might be into RKO cells via Lipofectamine 3000. GFP cells were collected by FACS 48 h after transfection. Cells were limiting diluted into 96-well plates. Cells were due to undescribed somatic mutations in these genetically unstable incubated at 37 °C in a CO incubator for 2–3 wk for single-clone generating. colon cancer cell lines. 2 It is now becoming clear that PRDM1 plays an important role Library Construction and RNA-Seq. RNA was extracted from RKO derivatives as a tumor suppressor in B, T, and lymphomas using the miRNeasy Mini Kit (catalog no. 217004; Qiagen), and RNA-seq libraries (23, 42). In colorectal cancers, PRDM1β expression levels vary in were constructed using TruSeq Stranded mRNA LT (catalog no. RS-122-2101; primary tumors and are correlated with patient disease-free Illumina) according to the manufacturers’ protocols. The DNA libraries were survival (Fig. 1G). Primary tumor cells that give rise to metas- qualified on an Agilent Technologies 2100 Bioanalyzer using an Agilent DNA tases often have molecular mechanisms and functional capabil- 1000 chip and were quantitated by qPCR in a Bio-Rad iCycler using a Bio-Rad ities similar to those in normal stem cells. Previous studies show iCycler qPCR Master Mix (catalog no. KK4844; Kapa Biosystems). After de- that breast cancers correlate with stem cell transcriptional sig- naturing, libraries were diluted to 1.8 pM with hybridization buffer. Paired- end 75-bp sequencing was performed on the Illumina NextSeq 500 benchtop natures in the context of p53 inactivation (37). Our RNA-seq sequencer using the NextSeq 500 High-output Kit v2 (150 CYS; catalog no. FC- results indicated that PRDM1 inhibits the expression of genes 404-2002; Illumina) per the manufacturer’sprotocol. that correlate with the stem cell transcriptional signature in RKO cells. Recently a long-term culture system has been developed RNA-Seq Analysis. Sequencing reads were mapped to the that allows the in vitro expansion and study of normal and tumor (hg19) using TopHat. Uniquely mapped reads were assembled into transcripts epithelial cells from a variety of epithelial tissues. These cultures, guided by the University of California, Santa Cruz (UCSC) Homo sapiens hg19 referred to as “organoids,” contain heterogeneous cell pop- RefSeq and Gencode gene annotation. Expression differences between ulations that derive from the normal stem cell compartment and conditions were evaluated using DESeq2 (43, 44). Pearson’s coefficient was reflect the in vivo cell-type diversity. We showed that p53 acti- calculated using the cor function with default parameters in R (https://www. vation increased PRDM1β expression in normal colon organo- r-project.org/). The hierarchical clustering analysis of the global gene- expression pattern in different samples was carried out using the ids. Moreover, we adapted the culture conditions for the long- heatmap.2 function (gplots package) in R. Gene set enrichment was ana- term expansion of colon tumor organoids and studied the effects lyzed with GSEA software (35). RNA-seq data have been deposited in the of PRDM1β overexpression on colony-formation ability and Gene Expression Omnibus (GEO) database (accession no. GSE101668). stem cell expansion. The two organoids lines we used in this study are TP53-null organoids. These assays showed a strong Human Tissue Processing and Crypt Isolation. Colonic tissues were obtained negative selection pressure with progressive elimination of from the University of Alabama at Birmingham Hospital with informed PRDM1-expressing cells. These findings are consistent with consent. All patients were diagnosed with colorectal cancer. From the studies in natural killer cell lymphoma (23) and are likely due to resected colon segment, normal as well as tumor tissue was isolated. The PRDM1 silencing the cancer stem cell core gene network (Fig. isolation of normal crypts and tumor epithelium was performed as described – 6B). Additionally, we found that PRDM1β is downstream of (28 30, 45). p53 protein in normal organoids, implying that p53 may elimi- For normal colon crypts isolation, human colon tissues were cut into small pieces. These pieces were washed three times with ice-cold Dulbecco’s PBS nate abnormal epithelial stem cells through PRDM1 during (DPBS) and then were incubated in cold DPBS supplemented with 2.5 mM cancer stem cell development by silencing stem cell genes. The EDTA for 40 min at 4 °C with gentle rotation. Then tubes were shaken mechanisms underlying the regulation of colon cancer stem cells vigorously to release crypts. The supernatant containing crypts was centri- and the possible effects on normal colon stem cell biology need fuged. The crypts were subsequently washed twice with DPBS and resus- to be addressed further. pended in Matrigel with about 100–200 crypts per 50 μL of Matrigel. Fifty In conclusion, our studies indicate that PRDM1β acts as a microliters of the crypt–Matrigel suspension were dispensed into the center tumor-suppressor gene in colon tumor organoids, likely by acting of each well of a 37 °C prewarmed 24-well plate. The Matrigel was poly- downstream of p53. It modifies the expression of stem cell- merized for 10 min at 37 °C. Human intestinal stem cell (HISC) medium was related genes, inhibits colon epithelial cell proliferation, and is added, and the crypts were incubated at 37 °C, 5% CO2 until they were ready to split. The composition of HISC medium was advanced DMEM/F12, positively associated with good survival in colon cancer patients. μ β 100 U/mL penicillin, 100 g/mL streptomycin, 2 mM GlutaMAX, 10 mM Since PRDM1 exerts a role in human colon tumor organoids, it Hepes, 1× N2, 1× B27 (Invitrogen), 1 mM N-acetyl-cysteine (Sigma), 10 mM may be a viable target for therapeutic strategies aimed at reac- Gastrin (Sigma) I, 50% Wnt-conditioned medium, 10% R-Spondin–condi- tivating the expression of PRDM1β in colon cancer cells. tioned medium, 100 ng noggin (Peprotech), 10 mM nicotinamide (Sigma), 50 ng/mL human EGF (Invitrogen), 500 nM A83-01 (Sigma), and 3 μM Materials and Methods SB202190 (Sigma) (SI Appendix, Table S2). Cell Culture. Human colon cancer RKO cells (kindly given by Bert Vogelstein, For tumor epithelium isolation, tumor tissues were cut into small pieces Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, and incubated in DPBS with collagenase/hyaluronidase (catalog no. 07919;

Baltimore) and its derivatives were maintained at 37 °C, 5% CO2 in McCoy’s STEMCELL Technology; final concentration: 2 mg/mL collagenase, 200 U/mL 5A medium (Fisher) supplemented with 10% FBS, 100 U/mL penicillin, and hyaluronidase) and 10 mM ROCK inhibitor for 30 min at 37 °C with shaking. 100 μg/mL streptomycin (Gibco). All cell lines were passaged in our labora- After incubation, heat-inactivated FBS (5%) was added, and the mixture was tory for no more than 40 passages after resuscitation. Cells were passaged allowed to sit for 2 min. Then the supernatant was transferred to a new tube every 3–4 d to maintain subconfluence. Authentication of the RKO cell line to remove large fragments. Cells were subsequently spun at 350 × g for was performed by Johns Hopkins University-Genetic Resources Core Facility 3 min. The pellet was washed twice in DBPS to remove debris and collage- Biorepository and Cell Center. nase. The tumor epithelium was resuspended in Matrigel and plated at

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Fig. 6. PRDM1 results in the down-regulation of the embryonic stem cell signature in cancer cells and the intestinal stem cell signature in normal intestinal stem cells. (A) GSEA of the embryonic stem cell signature and intestinal stem cell signature. ESC_EXPRESSION_1 contains 380 genes from Ben-Porath et al. (38); INTESTINAL_STEM_CELL_SIGNATURE contains 511 genes from Merlos-Suárez et al. (39). (B) A model illustrating the regulation of colon cancer cell pro- liferation by PRDM1 via multiple genes.

different densities. After the Matrigel was allowed to solidify, HISC medium solidify, HISC medium (for normal organoids) or HISC medium minus Wnt

without Wnt3a was added, and the cells were incubated at 37 °C, 5% CO2. (for tumor organoids), both supplemented with 10 μM ROCK inhibitor, was

added to the plates, and organoids were incubated at 37 °C, 5% CO2. Organoid Culture. The tumor and normal colon organoid culture medium was refreshed every 2 d. To passage the organoids, Matrigel was broken up with Tumor Organoid-Forming Assay. medium using a 1-mL pipette tip and was transferred from the well to a 15-mL Infection. The tumor organoids were cultured in 3D Matrigel culture. The tube. The organoids were centrifuged at 400 × g for 4 min, and the medium Matrigel was disrupted by pipetting, and organoids were collected into a was removed. One milliliter of TrypLE Selection (Invitrogen) was added, and 15-mL tube. The organoids were centrifuged at 400 × g for 4 min, and the the organoids were incubated at 37 °C for ∼5 min. Basal medium (Advanced supernatant was removed. Five microliters of TrypLE Selection (Invitrogen) DMEM/F12, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM GlutaMAX, were added, and the organoids were incubated at 37 °C for 20–40 min. Every 10 mM Hepes) was added, and cells were spun down at 400 × g for 4 min. 5 min organoids were fractured by up-and-down pipetting with 1-mL tips. The pellet was resuspended in Matrigel, and cells were plated in droplets of When most of the organoids were digested to single cells, basal medium 50 μL each in each well of 24-well plates. After the Matrigel was allowed to (Advanced DMEM/F12, 2 mM GlutaMAX, 10 mM Hepes, 1× penicillin/streptomycin)

Liu et al. PNAS Latest Articles | 9of10 Downloaded by guest on September 28, 2021 was added, and cells were spun down at 400 × g for 4 min. Cells were control sgRNA (catalog no. TELA1015) were purchased from transOMIC counted and resuspended in 500 μL of culture medium with 3 MOI of Technologies. Lentiviruses were packaged, concentrated, and titered. Tumor PRDM1β or control virus, 6 μg/mL Polybrene (Santa Cruz Biotechnology), and organoids were collected and digested with TrypLE to single cells. Cells were 10 μM ROCK inhibitor. Cells were transferred into one well of a 24-well plate infected with 1 MOI of Cas9 virus and 1 MOI of PRDM1 sgRNA virus or control and were incubated at 37 °C 5% CO2. After 6 h incubation, cells were col- sgRNA virus. After infection, cells were cultured in Matrigel with tumor HISC lected into a 1.5-mL tube and were centrifuged at 400 × g for 4 min. Cells medium with Blasticidin S and puromycin. The positive organoids were were resuspended in Matrigel and were replated in 50-μL droplets in each cultured and then were used for the organoid-forming assays. well of 24-well plates. Cell sorting and organoid formation. Cell sorting was done at days 2, 10, and Statistical Analysis. All data were analyzed with GraphPad Prism 6 (GraphPad 20 after virus transduction. Briefly, organoids were digested with TrypLE to ± + − Software) and are provided as mean SEM unless otherwise indicated. single cells as described above. Both GFP and GFP cells were collected by Statistical analyses were performed using an unpaired Student’s t test. The μ FACS. Five hundred single cells were resuspended in 50 L of Matrigel and significance level (P value) was set at *P < 0.05, **P < 0.01, and ***P < 0.001. replated in one well of 24-well plates. These cells were cultured for 3–5 wk. + The percentage of GFP cells was determined during cell sorting. ACKNOWLEDGMENTS. We thank Dr. Chang-uk Lim at the Microscopy and Flow Cytometry Core Facility of the College of Pharmacy at the University of PRDM1 KO in Human Colon Tumor Organoids. Lentiviral vectors for Cas9 South Carolina for cell sorting. This work was supported by NIH Grant (catalog no. TECC1002), PRDM1 sgRNA (catalog no. TEDH-1062730), and U01 CA158428 (to P.J.B.).

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